EP3679374B1 - Non-destructive sampling device for extraction of a marker - Google Patents

Non-destructive sampling device for extraction of a marker Download PDF

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Publication number
EP3679374B1
EP3679374B1 EP18726776.0A EP18726776A EP3679374B1 EP 3679374 B1 EP3679374 B1 EP 3679374B1 EP 18726776 A EP18726776 A EP 18726776A EP 3679374 B1 EP3679374 B1 EP 3679374B1
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Prior art keywords
sample
extraction
liquid
cell
housing
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German (de)
French (fr)
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EP3679374A1 (en
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Vincent Castronovo
Carl Emmerechts
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Universite de Liege ULG
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Universite de Liege ULG
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4005—Concentrating samples by transferring a selected component through a membrane
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids

Definitions

  • Non-destructive sample device for marker extraction for marker extraction.
  • the present invention relates to a method, device and non-destructive sample apparatus for extracting markers from a sample. More particularly the invention relates to a method, a device and an apparatus for the extraction of biomarkers from biological samples and its use for the diagnosis at the early stage of a disease.
  • Extracting low concentration markers from a sample is problematic when sample integrity must be maintained.
  • biomarkers or biological markers capable of detecting and evaluating the early stage of a disease such as cancer, or predicting resistance and response to treatment remains a challenge.
  • biomarkers are diluted up to a billion times from the concentration in diseased tissue making their detection from these media virtually impossible.
  • Gross et al. describes a method and device for the extraction of biomarkers from a sample which is practiced by subjecting the sample to a very high pressure variation at cycles of 20 seconds.
  • tissue samples such as solid biopsies
  • this precious human material is not accessible for the search for new useful and relevant biomarkers. These are intended in their integrity for complete histological evaluation in clinical testing. These samples are also taken in very small quantities, of the order of 1 to 125 mm 3 .
  • the detection of biomarkers is however essential to be able to make an individualized diagnosis and to allow the choice of the best possible therapy.
  • the aim of the present invention is to remedy a problem of low availability of markers during their extraction from samples of any origin whatsoever, when the integrity of the sample must be respected.
  • the aim of the present invention is to remedy the problem encountered when using a liquid biopsy such as blood, urine or saliva with too low a concentration of biomarkers, but also to remedy a lack of availability of solid biopsy.
  • a pressure cycle means that a pressure alternating between maximum and minimum pressure is applied for a determined time at a determined frequency.
  • sample any type of sample in the solid state, derived from food, plant tissue, biological tissue, mineral composition or other, likely to include one or more markers in low concentration in the sample.
  • the sample of biological tissues or biological sample generally comes from a surgical sample also called a biopsy, carried out for example by means of a needle or a scalpel.
  • Tissue removal can take place from any organ or part of the human or animal body such as, for example, the lung, breast, liver, kidney, uterus, prostate, bone marrow, muscle, colon, bronchus and the like.
  • the tissue sample may be from a cancerous tumor.
  • liquid is understood to mean any medium of low viscosity or of a viscosity close to water capable of dissolving markers present in a solid sample immersed in this medium.
  • liquid is meant, for example, a medium based on water, alcohol, oil and others.
  • An example of an aqueous medium used for a biological sample is physiological liquid.
  • physiological liquid a liquid isotonic with blood or exhibiting the same osmolarity as the main bodily fluids such as blood, urine and saliva.
  • the physiological liquid is for example composed of distilled water, of sodium chloride diluted to 0.9% of mass / volume of NaCl, ie 9 g / L.
  • the physiological liquid can be more complex and comprise, for example, potassium chloride (KCI), Calcium chloride (Ca Cl 2 ), Magnesium sulfate (MgSO 4 ) depending on the tissue considered.
  • phosphate buffered saline is also used with NaCl 9 g / L or other salts such as KCl or CaCl 2 as physiological liquid, particularly with samples from cancerous tumors.
  • the pressure cycle on the membrane of the extraction device must make it possible to obtain from the liquid, in particular physiological liquid, a sufficient quantity of markers, in particular biomarkers, extracted from the sample while avoiding the degradation of the sample, in particular the tissue sample or biopsy.
  • the application of a low pressure of between 2 and 5 bar, on the flexible membrane separating the liquid allows an extrusion from the sample of markers present in the sample.
  • the application of a low pressure of between 2 and 5 bar, on the flexible membrane separating the liquid allows extrusion from the tissue sample of interstitial fluid to cells of the tissue in which the biomarkers of interest are found.
  • the pressure cycle must nevertheless allow the morphology and antigenicity of the tissue sample to be maintained for the purposes of subsequent analyzes.
  • the pressure is reduced to atmospheric pressure and the cell is disconnected from the piston cylinder.
  • a fraction or all of the liquid, in particular physiological liquid, is extracted from the cell for detection of markers or biomarkers, while the basket with the sample is removed from the cell to allow further analyzes on the sample. having maintained its initial integrity. In the case of biological tissue samples, these will be subjected to subsequent clinical analyzes.
  • marker is understood to mean any substance present in low concentration in the solid sample and capable of dissolving in the sample immersion liquid.
  • a marker can be, for example, a biological compound, toxic or not present in a sample of animal or vegetable origin or a solid food.
  • a marker can also be a biomarker.
  • biomarker is meant not only proteins, but also circulating DNA, exosomes, mi RNAs, metabolites and equivalent molecules found in the interstitial fluid of the cells of the tissue in question.
  • the method according to the invention has the advantage that the sample is recovered with its morphological and chemical or biochemical integrity after extraction and detection of the markers or biomarkers.
  • the method of extracting markers or biomarkers has the advantage of being easy and rapid. It only takes a few minutes and does not cause any delay in subsequent testing on the sample such as clinical testing in the case of biological tissue samples. If part of the physiological liquid is kept in the extraction cell, it can be stored at low temperature in a cryopreservation liquid such as liquid nitrogen.
  • the extraction device (1) comprises an extraction cell (3) provided with a housing (12) for receiving a removable basket (5), said basket (5) being configured to immerse a sample (6) in a liquid previously introduced into the housing (12);
  • Each element belonging to the extraction device is manufactured by injection molding.
  • manufacturing is carried out in a clean room in order to meet the conditions for manufacturing a medical device.
  • the elements of the extraction device are washed with an organic solvent such as isopropanol in an ultrasonic bath.
  • the sterilization level of the elements thus produced must comply with the DIN EN ISO 17665 standard for medical devices and be such that no trace of DNA is identifiable.
  • the extraction cell is preferably made of a transparent polymeric material such as polymethacrylate as illustrated in figure 7 .
  • the extraction device (1) also comprises a cavity (7) separated from the housing (12) of the extraction cell by means of a flexible membrane (8).
  • the assembly consisting of the cavity and the flexible membrane allows compression or expansion of the liquid and of the tissue sample present in the hermetically sealed extraction cell.
  • the volume of the cavity is between 3 and 10mL, preferably 4.5mL.
  • the membrane (8) is made of a flexible polymeric material resistant to deformation of 50% to 200% of its surface.
  • the membrane must resist deformation during the compression of the liquid or the expansion of the liquid during storage of the extraction cell (3) in liquid nitrogen for example.
  • the flexibility of the membrane allows a deformation rate of 1 to 2 times its surface.
  • the membrane is preferably made of silicone with a thickness which can vary from 50 to 1000 microns with a preference of 100 microns.
  • the flexible membrane preferably of silicone, is fixed between two assembly parts and is arranged for example at the base of the extraction cell (3), by means of a V-shaped groove.
  • the two assembly parts can optionally be separated.
  • the extraction device (1) as shown in figure 3 also comprises a sealed cap, arranged on the extraction cell (3) to fix the removable support basket (5) in the housing (12) and to maintain the liquid inside the housing (12) during the cleaning cycle. pressure.
  • the leaktight stopper comprises two parts, an upper part (2) or Luer Lock stopper arranged on a lower part (4) configured to allow the extraction of the liquid from the cell and to maintain the airtightness of the cell. extraction during the pressure cycle.
  • the lower part of the stopper is preferably screwed onto the extraction cell by means of a cone-to-cone system, thus making it possible to seal the extraction cell.
  • the support basket (5) is made by injection molding, also of plastic, preferably polypropylene. In a preferred embodiment the support basket is disposable.
  • the basket is provided with vertical ribs allowing the immersion of the tissue sample in physiological liquid.
  • the ribs of the container should be of a width suitable for the size of the sample to allow the sample to remain in the basket while being submerged in physiological liquid.
  • the volume of the support basket (5) in the housing (12) of the extraction cell (3) represents between 10 and 50%, preferably 20% of the volume of the housing inside the extraction cell. Basket volume is also related to sample size.
  • the extraction device comprises a cylinder (10) provided with a piston (11), said cylinder being connected to the cavity (9) at the orifice (13).
  • the cavity-membrane assembly provides a uniform distribution of the pressure on the liquid and a better performance of extraction of the markers
  • the volume of the cavity and the cylinder above the piston is filled with air, but any other inert gas can also be used such as nitrogen, argon or the like.
  • the total volume of the cavity and the air-filled cylinder may vary from 20 to 100 ml, but is preferably of the order of 30 ml.
  • the cylinder provided with the piston is a disposable medical syringe.
  • the cylinder is generally connected vertically to the extrusion cell, but any other configuration, such as for example a horizontal configuration is also possible.
  • the cavity comprises one or more stiffeners (9).
  • the stiffeners are a multiple of 2, they are preferably arranged in a star pattern around the connection hole (13) of the cylinder (10).
  • the use of a stiffener advantageously makes it possible to avoid sticking of the membrane to the wall of the cavity.
  • the liquid will preferably be a physiological liquid for the extraction of biomarkers from a sample of biological tissues immersed in this physiological liquid.
  • the present invention also relates to a 3 rd aspect, a biomarker extraction device for early diagnosis of diseases at an early stage or for treatment of disease, especially for the treatment of cancer.
  • the present invention also relates in a 4 th aspect, a use of the device for the detection of protein biomarkers, molecular metabolic from tissue sample or solid biopsy.
  • the present invention relates to a 5 th aspect, an apparatus as illustrated in figure 6 integrating the extraction device as well as means for actuating the pressure piston such as a cylinder.
  • the apparatus may also include an automatic parameter control module.
  • the figure 7 shows an example of an apparatus comprising the extraction cell as well as a 30mL surgical syringe as a piston cylinder.
  • the method, device and apparatus have been tested on a large series of biological tissue samples obtained from primary colorectal tumors (CRC) and liver metastasis (CRC-LM).
  • CRC primary colorectal tumors
  • CRC-LM liver metastasis
  • Freshly surgically taken biopsies were collected immediately after the operation, cut into a 3 mm 3 sample and placed in a 4.5 ml cell.
  • the syringe plunger is then moved to the 15mL mark.
  • the syringe is then connected to the extraction cell.
  • the sample is then subjected to alternating pressure at a frequency of 1 Hz for one minute by moving the plunger in the syringe between the 15mL marking and 7.5 mL.
  • tissue samples were subjected to the method.
  • Each tissue sample was divided in half; on the one hand for routine pathological analysis and on the other hand for the extraction method according to the invention also called INV in the figures 8 and 9 .
  • the samples for the routine analysis and for the extraction method according to the invention are subjected to the same immunohistochemistry (IHC) according to the protocol known to those skilled in the art and described by A.Bellahcene and V. Castronovo in Am. J Pathol 1955 Jan; 146 (1): 95-100 .
  • IHC immunohistochemistry
  • H&E hematoxylin / eosin
  • Example 2 Ten breast cancer samples and their neighboring non-tumor tissue were treated according to the protocol described in Example 1 and then subjected to the device and method according to the invention.
  • the liquid obtained after application of the extraction device was then analyzed by mass spectrometry for the detection of soluble proteins and by Nuclear Magnetic Resonance for the detection of metabolites.
  • the markers identified from the fluid obtained from cancerous tissue were then compared with those from normal tissue.
  • Proteins detected in cancerous tissues for example, Periostin, comp, Ezrin, Raxidin, Versican, Tenascin are identified. These proteins are generally present in the extracellular space of tissues as illustrated in Table 1 below, showing extracts from Pubmed: Table 1: Proteins Identified in Breast Cancer Tissues Periostin 49 Results on Pubmed COMP. 4 results on Pubmed • Protein involved in osteoblast adhesion. • Localization: extracellular space • Major component of ECM. • Tumor development • Secreted by fibroblasts • Dissemination metastases • Generally associated with poor patient outcome.
  • Ezrin 114 results on Pubmed Radixin 63 results on Pubmed • Localization: plasma membrane Major association: • Localization: cell membrane, extracellular space • Ezrin-radixin-moesin (ERM) plys important role in the maintenance of cytoskeleton structure and cells movement. It also involved in breast cancer invasion and metastasis and may be a potential biomarker.
  • EEM Ezrin-radixin-moesin
  • the figure 10 illustrates the number of protein markers identified in the liquid obtained following the device and the extraction method according to the invention.
  • the markers identified from the liquid in which cancerous tissue has been immersed are compared with those extracted from normal tissue.
  • the Ingenuity Patway Analysis was used for the interpretation of the collected data and their analysis using the IPA Core Analysis software.
  • the value of P indicates which molecule is significantly associated with the introduced reference molecules and this relatively to all the functional molecules characterized.
  • the fold enrichment indicates the statistical significance of the presence of the metabolite in the sample.

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Description

Dispositif non-destructif d'échantillon pour extraction de marqueur.Non-destructive sample device for marker extraction.

La présente invention concerne une méthode, un dispositif et un appareillage non destructif d'échantillon, pour l'extraction de marqueurs à partir d'un échantillon. Plus particulièrement l'invention concerne une méthode, un dispositif et un appareillage pour l'extraction de biomarqueurs à partir d'échantillons biologiques et son utilisation pour le diagnostic à l'état précoce d'une maladie.The present invention relates to a method, device and non-destructive sample apparatus for extracting markers from a sample. More particularly the invention relates to a method, a device and an apparatus for the extraction of biomarkers from biological samples and its use for the diagnosis at the early stage of a disease.

L'extraction de marqueurs en faible concentration dans un échantillon, quel que soit son origine se révèle problématique lorsque l'intégrité de l'échantillon doit être maintenu.Extracting low concentration markers from a sample, regardless of its origin, is problematic when sample integrity must be maintained.

En particulier, La recherche de biomarqueurs ou marqueurs biologiques capables de détecter et d'évaluer le stade précoce d'une maladie tel que le cancer, ou prédire la résistance et la réponse au traitement reste un challenge.In particular, the search for biomarkers or biological markers capable of detecting and evaluating the early stage of a disease such as cancer, or predicting resistance and response to treatment remains a challenge.

Actuellement, la recherche non invasive de biomarqueurs se fait essentiellement au départ d'échantillons de sang, de sérum, d'urine ou de salive. Dans ces liquides, les biomarqueurs sont dilués jusqu'à un milliard de fois par rapport à la concentration au niveau du tissu malade ce qui rend leur détection à partir de ces milieux virtuellement impossible. Gross et al. (Journal of biomolecular techniques, 2008 ) décrit une méthode et un dispositif pour l'extraction de biomarqueurs d'un échantillon qui est pratiqué en soumettant l'échantillon à une variation de pression très haute à de cycles de 20 secondes.Currently, non-invasive research for biomarkers is mainly carried out using samples of blood, serum, urine or saliva. In these fluids, biomarkers are diluted up to a billion times from the concentration in diseased tissue making their detection from these media virtually impossible. Gross et al. (Journal of biomolecular techniques, 2008 ) describes a method and device for the extraction of biomarkers from a sample which is practiced by subjecting the sample to a very high pressure variation at cycles of 20 seconds.

La deuxième limitation est l'inaccessibilité d'échantillon de tissus tels que les biopsies solides, prioritairement destinés dans leur totalité à l'analyse pour du diagnostic. Ainsi, pour des raisons éthiques évidentes, ce matériel humain précieux n'est pas accessible pour la recherche de nouveaux biomarqueurs utiles et pertinents. Ceux-ci sont destinés dans leur intégrité pour l'évaluation histologique complète en test clinique. Ces échantillons sont d'ailleurs prélevés en quantité très faibles, de l'ordre de 1 à 125 mm3.The second limitation is the inaccessibility of tissue samples such as solid biopsies, primarily intended in their entirety for analysis for diagnosis. Thus, for obvious ethical reasons, this precious human material is not accessible for the search for new useful and relevant biomarkers. These are intended in their integrity for complete histological evaluation in clinical testing. These samples are also taken in very small quantities, of the order of 1 to 125 mm 3 .

La détection de biomarqueurs est pourtant indispensable pour pouvoir poser un diagnostic individualisé et permettre le choix de la meilleure thérapie possible. Le but de la présente invention est de remédier à un problème de faible disponibilité de marqueurs lors de leur extraction à partir d'échantillons de quelque origine que ce soit, lorsque l'intégrité de l'échantillon doit être respectée.The detection of biomarkers is however essential to be able to make an individualized diagnosis and to allow the choice of the best possible therapy. The aim of the present invention is to remedy a problem of low availability of markers during their extraction from samples of any origin whatsoever, when the integrity of the sample must be respected.

Plus particulièrement, le but de la présente invention est de remédier au problème rencontré lors de l'utilisation de biopsie liquide telle que le sang, l'urine ou la salive avec une concentration trop faible en biomarqueurs, mais également de remédier à un manque de disponibilité de biopsie solide.More particularly, the aim of the present invention is to remedy the problem encountered when using a liquid biopsy such as blood, urine or saliva with too low a concentration of biomarkers, but also to remedy a lack of availability of solid biopsy.

L'invention concerne dans un premier aspect une méthode aisée et rapide d'extraction de marqueur à partir d'échantillon comprenant les étapes suivantes:

  • introduction d'un échantillon dans un panier-support jetable placé dans le logement d'une cellule d'un dispositif d'extraction muni d'une membrane flexible et
  • immersion de l'échantillon dans un liquide préalablement introduit dans le logement de la cellule;
  • fermeture hermétique de la cellule d'extraction après immersion de l'échantillon dans le liquide et application d'un cycle de pression sur la membrane flexible pendant une durée de 1 à 5 minutes, de préférence 3 minutes ;à une fréquence comprise entre 1 et 2 Hz, de préférence 1,5 Hz ;puis
  • récupération totale ou partielle du liquide immergeant l'échantillon après application du cycle de pression sur la membrane et détection des marqueurs présents dans le liquide.
la pression hydrostatique appliquée sur la membrane du dispositif est comprise entre 2 et 5 bar, et est de préférence 2 bar.The invention relates in a first aspect to an easy and rapid method of extracting a label from a sample comprising the following steps:
  • introduction of a sample into a disposable support basket placed in the housing of a cell of an extraction device fitted with a flexible membrane and
  • immersion of the sample in a liquid previously introduced into the housing of the cell;
  • hermetic closure of the extraction cell after immersing the sample in the liquid and applying a pressure cycle to the flexible membrane for a period of 1 to 5 minutes, preferably 3 minutes; at a frequency between 1 and 2 Hz, preferably 1.5 Hz; then
  • total or partial recovery of the liquid immersing the sample after application of the pressure cycle on the membrane and detection of the markers present in the liquid.
the hydrostatic pressure applied to the membrane of the device is between 2 and 5 bar, and is preferably 2 bar.

Un cycle de pression signifie qu'une pression alternant entre maximum et minimum de pression est appliquée pendant un temps déterminé à une fréquence déterminée.A pressure cycle means that a pressure alternating between maximum and minimum pressure is applied for a determined time at a determined frequency.

Par échantillon on entend tout type d'échantillon à l'état solide, issu d'aliments, de tissus végétal, de tissus biologiques, de composition minérale ou autre, susceptibles de comporter un ou plusieurs marqueurs en faible concentration dans l'échantillon. Plus particulièrement, l'échantillon de tissus biologique ou échantillon biologique est généralement issu d'un prélèvement chirurgical aussi dénommé biopsie, effectué par exemple au moyen d'une aiguille ou d'un bistouri. Le prélèvement de tissus peut avoir lieu au niveau de n'importe quel organe ou élément du corps humain ou animal comme par exemple du poumon, sein, foie, rein, utérus, prostate, moelle osseuse, muscle, colon, bronche et autre. L'échantillon de tissu peut être issu d'une tumeur cancéreuse.By sample is meant any type of sample in the solid state, derived from food, plant tissue, biological tissue, mineral composition or other, likely to include one or more markers in low concentration in the sample. More particularly, the sample of biological tissues or biological sample generally comes from a surgical sample also called a biopsy, carried out for example by means of a needle or a scalpel. Tissue removal can take place from any organ or part of the human or animal body such as, for example, the lung, breast, liver, kidney, uterus, prostate, bone marrow, muscle, colon, bronchus and the like. The tissue sample may be from a cancerous tumor.

Par liquide, on entend tout milieu de faible viscosité ou de viscosité proche de l'eau susceptible de solubiliser des marqueurs présents dans un échantillon solide immergé dans ce milieu. Par liquide on entend par exemple un milieu à base d'eau, d'alcool, d'huile et autres. Un exemple de milieu aqueux utilisé pour un échantillon biologique est un liquide physiologique.The term liquid is understood to mean any medium of low viscosity or of a viscosity close to water capable of dissolving markers present in a solid sample immersed in this medium. By liquid is meant, for example, a medium based on water, alcohol, oil and others. An example of an aqueous medium used for a biological sample is physiological liquid.

Par liquide physiologique, on entend un liquide isotonique au sang ou présentant la même osmolarité que les principaux fluides corporels tels que le sang, l'urine, la salive. le liquide physiologique est par exemple composé d'eau distillée, de chlorure de sodium dilué à 0,9% de masse/volume de NaCl, soit 9 g/L. le liquide physiologique peut être plus complexe et comprendre par exemple du chlorure de potassium (KCI), chlorure de Calcium (Ca Cl2), sulfate de Magnésium (MgSO4) selon le tissu considéré.By physiological liquid is meant a liquid isotonic with blood or exhibiting the same osmolarity as the main bodily fluids such as blood, urine and saliva. the physiological liquid is for example composed of distilled water, of sodium chloride diluted to 0.9% of mass / volume of NaCl, ie 9 g / L. the physiological liquid can be more complex and comprise, for example, potassium chloride (KCI), Calcium chloride (Ca Cl 2 ), Magnesium sulfate (MgSO 4 ) depending on the tissue considered.

Enfin le tampon phosphate salin (PBS) est également utilisé avec NaCl 9 g/L ou autre sels tels que KCl ou CaCl2 en tant que liquide physiologique particulièrement avec des échantillons issus de tumeurs cancéreuses.Finally, phosphate buffered saline (PBS) is also used with NaCl 9 g / L or other salts such as KCl or CaCl 2 as physiological liquid, particularly with samples from cancerous tumors.

Le cycle de pression sur la membrane du dispositif d'extraction, doit permettre d'obtenir à partir du liquide, en particulier du liquide physiologique une quantité suffisante de marqueurs, en particulier des biomarqueurs, extrait de l'échantillon tout en évitant la dégradation de l'échantillon, en particulier l'échantillon de tissus ou biopsie. L'application d'une basse pression comprise entre 2 et 5 bar, sur la membrane flexible séparant le liquide permet une extrusion à partir de l'échantillon de marqueurs présents dans l'échantillon. Dans le cas d'un échantillon biologique, l'application d'une basse pression comprise entre 2 et 5 bar, sur la membrane flexible séparant le liquide permet une extrusion à partir de l'échantillon du tissu, d'un fluide interstitiel aux cellules du tissu dans lequel se trouvent les biomarqueurs d'intérêt. Le cycle de pression doit permettre néanmoins de maintenir la morphologie et l'antigènicité de l'échantillon de tissus aux fins d'analyses ultérieures.The pressure cycle on the membrane of the extraction device must make it possible to obtain from the liquid, in particular physiological liquid, a sufficient quantity of markers, in particular biomarkers, extracted from the sample while avoiding the degradation of the sample, in particular the tissue sample or biopsy. The application of a low pressure of between 2 and 5 bar, on the flexible membrane separating the liquid allows an extrusion from the sample of markers present in the sample. In the case of a biological sample, the application of a low pressure of between 2 and 5 bar, on the flexible membrane separating the liquid allows extrusion from the tissue sample of interstitial fluid to cells of the tissue in which the biomarkers of interest are found. The pressure cycle must nevertheless allow the morphology and antigenicity of the tissue sample to be maintained for the purposes of subsequent analyzes.

Après avoir été soumise à une pression cyclique pendant une courte durée de 1 à 5 minutes, la pression est ramenée à pression atmosphérique et la cellule est déconnectée du cylindre à piston. Une fraction ou la totalité du liquide, en particulier le liquide physiologique est extrait de la cellule pour détection de marqueurs ou biomarqueurs, tandis que le panier muni de l'échantillon est retiré de la cellule pour permettre d'autres analyses ultérieures sur l'échantillon ayant maintenu son intégrité initiale. Dans le cas d'échantillon de tissus biologiques, ceux-ci seront soumis à des analyses cliniques ultérieures.After being subjected to cyclic pressure for a short period of 1 to 5 minutes, the pressure is reduced to atmospheric pressure and the cell is disconnected from the piston cylinder. A fraction or all of the liquid, in particular physiological liquid, is extracted from the cell for detection of markers or biomarkers, while the basket with the sample is removed from the cell to allow further analyzes on the sample. having maintained its initial integrity. In the case of biological tissue samples, these will be subjected to subsequent clinical analyzes.

Par marqueur, on entend toute substance présente en faible concentration dans l'échantillon solide et susceptible de se solubiliser dans le liquide d'immersion de l'échantillon. Un marqueur peut être, par exemple, un composé biologique, toxique ou non présent dans un échantillon d'origine animale, végétale ou un aliment solide. Un marqueur peut également être un biomarqueur.The term “marker” is understood to mean any substance present in low concentration in the solid sample and capable of dissolving in the sample immersion liquid. A marker can be, for example, a biological compound, toxic or not present in a sample of animal or vegetable origin or a solid food. A marker can also be a biomarker.

Par biomarqueur, on entend non seulement des protéines, mais également du DNA circulant, des exosomes, mi RNA, métabolites et molécules équivalentes se trouvant dans le liquide interstitiel aux cellules du tissu considéré.By biomarker is meant not only proteins, but also circulating DNA, exosomes, mi RNAs, metabolites and equivalent molecules found in the interstitial fluid of the cells of the tissue in question.

La méthode selon l'invention présente l'avantage que l'échantillon est récupéré avec son intégrité morphologique et chimique ou biochimique après extraction et détection des marqueurs ou biomarqueurs.The method according to the invention has the advantage that the sample is recovered with its morphological and chemical or biochemical integrity after extraction and detection of the markers or biomarkers.

La méthode d'extraction de marqueurs ou biomarqueurs a l'avantage d'être aisée et rapide. Elle ne prend que quelques minutes et n'occasionne aucun retard aux tests ultérieurs sur l'échantillon comme les tests cliniques dans le cas d'échantillons de tissus biologiques. Si une partie du liquide physiologique est maintenu dans la cellule d'extraction, il peut être stocké à basse température dans un liquide de cryopréservation comme l'azote liquide.The method of extracting markers or biomarkers has the advantage of being easy and rapid. It only takes a few minutes and does not cause any delay in subsequent testing on the sample such as clinical testing in the case of biological tissue samples. If part of the physiological liquid is kept in the extraction cell, it can be stored at low temperature in a cryopreservation liquid such as liquid nitrogen.

L'invention concerne dans un deuxième aspect, un dispositif pour l'extraction de marqueur qui pour l'intelligence de l'invention est illustré dans les figures 1 à 7.

  • Figure 1 : schéma de la cellule d'extraction selon l'invention
  • Figure 2 : étape d'introduction de l'échantillon dans la cellule d'extraction
  • Figure 3 : étape de fermeture hermétique de la cellule d'extraction au moyen d'un bouchon constitué de deux parties.
  • Figure 4 : illustration du système de pression sur le liquide dans lequel est plongé l'échantillon.
  • Figure 5 : étape de retrait après le cycle de pression, du panier-support comprenant l'échantillon.
  • Figure 6 : schéma de l'appareillage comprenant le dispositif d'extraction selon l'invention
  • Figure 7 : exemple de cellule d'extraction et d'appareillage selon l'invention.
  • Figure 8 et 9 comparaison analyse Immunohistochimie (IHC) sur échantillons de tissus biologiques ayant subi l'extraction de biomarqueurs selon invention et échantillons de référence non soumis à l'extraction.
  • Figure 10 : exemple d'utilisation du dispositif selon l'invention pour la détection de marqueurs protéiques.
  • Figure 11 : exemple d'utilisation du dispositif selon l'invention pour la détection de marqueurs métaboliques
The invention relates in a second aspect, a device for the extraction of marker which for the intelligence of the invention is illustrated in the figures 1 to 7 .
  • Figure 1 : diagram of the extraction cell according to the invention
  • Figure 2 : step of introducing the sample into the extraction cell
  • Figure 3 : step of hermetically closing the extraction cell by means of a stopper made up of two parts.
  • Figure 4 : illustration of the pressure system on the liquid in which the sample is immersed.
  • Figure 5 : step of withdrawal after the pressure cycle, of the support basket comprising the sample.
  • Figure 6 : diagram of the apparatus comprising the extraction device according to the invention
  • Figure 7 : example of an extraction cell and apparatus according to the invention.
  • Figure 8 and 9 comparison immunohistochemistry analysis (IHC) on samples of biological tissues having undergone the extraction of biomarkers according to the invention and reference samples not subjected to the extraction.
  • Figure 10 : example of use of the device according to the invention for the detection of protein markers.
  • Figure 11 : example of use of the device according to the invention for the detection of metabolic markers

Les figures servent à illustrer les modes de réalisation de l'invention, mais ne sont pas limitatives de l'invention.The figures serve to illustrate the embodiments of the invention, but are not limiting of the invention.

Le dispositif d'extraction (1) comprend une cellule d'extraction (3) muni d'un logement (12) pour recevoir un panier amovible (5), ledit panier (5) étant configuré pour immerger un échantillon (6) dans un liquide préalablement introduit dans le logement (12) ;
Chaque élément appartenant au dispositif d'extraction est fabriqué par moulage à injection.The extraction device (1) comprises an extraction cell (3) provided with a housing (12) for receiving a removable basket (5), said basket (5) being configured to immerse a sample (6) in a liquid previously introduced into the housing (12);
Each element belonging to the extraction device is manufactured by injection molding.

Dans le cas de dispositif à usage biologique, la fabrication est réalisée en salle blanche afin de répondre aux conditions de fabrication d'un dispositif médical. Une fois fabriqués, les éléments du dispositif d'extraction sont lavés au moyen d'un solvant organique tel que de l'isopropanol dans un bain ultrasonique. Le niveau de stérilisation des éléments ainsi fabriqué doit être conforme au standard DIN EN ISO 17665 pour dispositifs médicaux et être tel qu'aucune trace d'ADN ne soit identifiable.In the case of a device for biological use, manufacturing is carried out in a clean room in order to meet the conditions for manufacturing a medical device. Once manufactured, the elements of the extraction device are washed with an organic solvent such as isopropanol in an ultrasonic bath. The sterilization level of the elements thus produced must comply with the DIN EN ISO 17665 standard for medical devices and be such that no trace of DNA is identifiable.

La cellule d'extraction est de préférence fabriquée en un matériau polymérique transparent tel que le polyméthacrylate comme illustrée à la figure 7.The extraction cell is preferably made of a transparent polymeric material such as polymethacrylate as illustrated in figure 7 .

Le dispositif d'extraction (1) comprend également une cavité (7) séparée du logement (12) de la cellule d'extraction au moyen d'une membrane flexible (8).The extraction device (1) also comprises a cavity (7) separated from the housing (12) of the extraction cell by means of a flexible membrane (8).

L'ensemble constitué de la cavité et de la membrane flexible permet une compression ou une dilatation du liquide et de l'échantillon de tissus présent dans la cellule d'extraction hermétiquement fermée.The assembly consisting of the cavity and the flexible membrane allows compression or expansion of the liquid and of the tissue sample present in the hermetically sealed extraction cell.

Le volume de la cavité est compris entre 3 et 10mL, de préférence 4.5mL.The volume of the cavity is between 3 and 10mL, preferably 4.5mL.

La membrane (8) est en matériau polymérique flexible et résistant à une déformation de 50% à 200% de sa surface. La membrane doit résister à la déformation lors de la compression du liquide ou de la dilatation du liquide lors d'un stockage de la cellule d'extraction (3) dans l'azote liquide par exemple. De préférence, la flexibilité de la membrane permet un taux de déformation de 1 à 2 fois sa surface.The membrane (8) is made of a flexible polymeric material resistant to deformation of 50% to 200% of its surface. The membrane must resist deformation during the compression of the liquid or the expansion of the liquid during storage of the extraction cell (3) in liquid nitrogen for example. Preferably, the flexibility of the membrane allows a deformation rate of 1 to 2 times its surface.

La membrane est de préférence en silicone avec une épaisseur qui peut varier de 50 à 1000 micron avec une préférence de 100 microns.The membrane is preferably made of silicone with a thickness which can vary from 50 to 1000 microns with a preference of 100 microns.

La membrane flexible, de préférence en silicone, est fixée entre deux pièces d'assemblement et est agencée par exemple à la base de la cellule d'extraction (3), au moyen d'une rainure en V. Les deux pièces d'assemblement peuvent être optionnellement désolidarisées.The flexible membrane, preferably of silicone, is fixed between two assembly parts and is arranged for example at the base of the extraction cell (3), by means of a V-shaped groove. The two assembly parts can optionally be separated.

Le dispositif d'extraction (1) comme illustré à la figure 3, comprend également un bouchon étanche, agencé sur la cellule d'extraction (3) pour fixer le panier-support (5) amovible dans le logement(12) et maintenir le liquide à l'intérieur du logement (12) durant le cycle de pression.The extraction device (1) as shown in figure 3 , also comprises a sealed cap, arranged on the extraction cell (3) to fix the removable support basket (5) in the housing (12) and to maintain the liquid inside the housing (12) during the cleaning cycle. pressure.

Selon un mode préféré, le bouchon étanche comprend deux parties, une partie supérieure (2) ou bouchon Luer Lock agencé sur une partie inférieure (4) configurée pour permettre l'extraction du liquide de la cellule et pour maintenir l'étanchéité de la cellule d'extraction durant le cycle de pression.According to a preferred embodiment, the leaktight stopper comprises two parts, an upper part (2) or Luer Lock stopper arranged on a lower part (4) configured to allow the extraction of the liquid from the cell and to maintain the airtightness of the cell. extraction during the pressure cycle.

La partie inférieure du bouchon est de préférence vissée sur la cellule d'extraction au moyen d'un système cône à cône permettant ainsi l'étanchéité de la cellule d'extraction.The lower part of the stopper is preferably screwed onto the extraction cell by means of a cone-to-cone system, thus making it possible to seal the extraction cell.

Le panier-support (5) est fabriqué par moulage à injection, en matière plastique également, de préférence du polypropylène. Dans un mode de réalisation préféré le panier-support est jetable.The support basket (5) is made by injection molding, also of plastic, preferably polypropylene. In a preferred embodiment the support basket is disposable.

De préférence, le panier est muni de nervures verticales permettant l'immersion de l'échantillon de tissus dans le liquide physiologique. Les nervures du récipient doivent avoir une largeur adaptée à la taille de l'échantillon pour permettre à l'échantillon de rester dans le panier tout en étant immergé dans le liquide physiologique.Preferably, the basket is provided with vertical ribs allowing the immersion of the tissue sample in physiological liquid. The ribs of the container should be of a width suitable for the size of the sample to allow the sample to remain in the basket while being submerged in physiological liquid.

Le volume du panier-support (5) dans le logement (12) de la cellule d'extraction (3) représente entre 10 et 50%, de préférence 20% du volume du logement à l'intérieur de la cellule d'extraction. Le volume du panier est également lié à la taille de l'échantillon. Selon un autre mode de réalisation préféré, le dispositif d'extraction comprend un cylindre (10) muni d'un piston (11), ledit cylindre étant connecté à la cavité (9) à l'orifice (13). L'ensemble cavité-membrane permet d'obtenir une répartition uniforme de la pression sur le liquide et une meilleure performance d'extraction des marqueurs De préférence, le volume de la cavité et du cylindre au-dessus du piston est rempli d'air, mais tout autre gaz inerte peut également être utilisé tel que azote, argon ou équivalent. Le volume total de la cavité et du cylindre rempli d'air peut varier de 20 à 100 ml, mais est de préférence de l'ordre de 30 ml.The volume of the support basket (5) in the housing (12) of the extraction cell (3) represents between 10 and 50%, preferably 20% of the volume of the housing inside the extraction cell. Basket volume is also related to sample size. According to another preferred embodiment, the extraction device comprises a cylinder (10) provided with a piston (11), said cylinder being connected to the cavity (9) at the orifice (13). The cavity-membrane assembly provides a uniform distribution of the pressure on the liquid and a better performance of extraction of the markers Preferably, the volume of the cavity and the cylinder above the piston is filled with air, but any other inert gas can also be used such as nitrogen, argon or the like. The total volume of the cavity and the air-filled cylinder may vary from 20 to 100 ml, but is preferably of the order of 30 ml.

Dans un mode encore plus préféré du dispositif de l'invention, le cylindre muni du piston est une seringue médicale jetable.In an even more preferred embodiment of the device of the invention, the cylinder provided with the piston is a disposable medical syringe.

Le cylindre est généralement connecté verticalement à la cellule d'extrusion, mais tout autre configuration, comme par exemple une configuration horizontale est également possible.The cylinder is generally connected vertically to the extrusion cell, but any other configuration, such as for example a horizontal configuration is also possible.

Selon un autre mode de réalisation préféré la cavité comprend un ou plusieurs raidisseurs (9). Lorsque les raidisseurs sont un multiple de 2, ils sont de préférence disposés en étoile autour de l'orifice (13) de connexion du cylindre (10). L'utilisation de raidisseur permet d'éviter avantageusement un collement de la membrane à la paroi de la cavité.According to another preferred embodiment, the cavity comprises one or more stiffeners (9). When the stiffeners are a multiple of 2, they are preferably arranged in a star pattern around the connection hole (13) of the cylinder (10). The use of a stiffener advantageously makes it possible to avoid sticking of the membrane to the wall of the cavity.

Dans le cas de dispositif à usage biologique, le liquide sera de préférence un liquide physiologique pour l'extraction de biomarqueurs à partir d'un échantillon de tissus biologiques immergé dans ce liquide physiologique.In the case of a device for biological use, the liquid will preferably be a physiological liquid for the extraction of biomarkers from a sample of biological tissues immersed in this physiological liquid.

La présente invention concerne également dans un 3e aspect, un dispositif d'extraction de biomarqueurs pour diagnostic précoce de maladies à un stade primaire ou pour traitement de maladie, plus particulièrement pour le traitement de cancer.The present invention also relates to a 3 rd aspect, a biomarker extraction device for early diagnosis of diseases at an early stage or for treatment of disease, especially for the treatment of cancer.

La présente invention concerne également dans un 4e aspect, une utilisation du dispositif pour la détection de biomarqueurs protéiques, métabolique moléculaires à partir d'échantillon de tissus ou biopsie solide.The present invention also relates in a 4 th aspect, a use of the device for the detection of protein biomarkers, molecular metabolic from tissue sample or solid biopsy.

Enfin la présente invention concerne un 5e aspect, un appareillage comme illustré à la figure 6 intégrant le dispositif d'extraction ainsi que des moyens pour actionner le piston de pression comme par exemple un verrin. L'appareillage peut comprendre également un module automatique de contrôle de paramètres.Finally, the present invention relates to a 5 th aspect, an apparatus as illustrated in figure 6 integrating the extraction device as well as means for actuating the pressure piston such as a cylinder. The apparatus may also include an automatic parameter control module.

La figure 7 représente un exemple d'un appareillage comprenant la cellule d'extraction ainsi qu'une seringue chirurgicale de 30mL comme cylindre à piston.The figure 7 shows an example of an apparatus comprising the extraction cell as well as a 30mL surgical syringe as a piston cylinder.

ExemplesExamples

La méthode, le dispositif et l'appareillage ont été testés sur une grande série d'échantillons de tissus biologiques obtenus à partir de tumeurs colorectales primaires (CRC) et de métastase de foie (CRC-LM). Le liquide extrudé à partir des échantillons est un matériel unique pour la détection de biomarqueurs et n'interfère nullement dans la morphologie des tissus pour les tests cliniques ultérieurs comme illustré ci-après.The method, device and apparatus have been tested on a large series of biological tissue samples obtained from primary colorectal tumors (CRC) and liver metastasis (CRC-LM). The liquid extruded from the samples is a unique material for the detection of biomarkers and does not interfere with tissue morphology for subsequent clinical testing as illustrated below.

1.Application de la méthode d'extraction selon l'invention.1.Application of the extraction method according to the invention.

Des biopsies fraichement prélevées chirurgicalement ont été collectées immédiatement après opération, découpées en échantillon de 3 mm3 et placé dans une cellule de 4,5 mL. Quatre mL de tampon PBS hypertonic avec NaCl, 4.5 g dans 500 mL (Weestburg) a été ajouté dans la cellule en même temps que l'échantillon dans le panier. Indépendamment, le piston de la seringue est alors déplacé jusqu'au marquage de 15mL. La seringue est ensuite connectée à la cellule d'extraction. L'échantillon est ensuite soumis à une pression alternative à une fréquence de 1 Hz pendant une minute par le déplacement du piston dans la seringue entre le marquage 15mL et 7,5 mL. Lors de mesures effectuées séparément avec un manomètre connecté, il apparait que ce changement de volume correspond à une variation de pression effective entre 2 bar (état de compression) et 1 bar bar (état relaxé) et 1 bar au point initial lorsque le piston se trouve à la marque 15mL. La procédure est répétée 3 fois pour chaque exemple. Les fluides d'extraction ont ensuite été conservés à -20°C. Toute la procédure a duré 3 minutes et l'échantillon est restitué pour évaluation clinique. Un protocole semblable a été appliqué à des échantillons issus de tumeurs du cancer du sein humain, du cancer colorectal primaire et des métastases de foie et du colon ainsi que sur leur contreparties normales adjacentes.Freshly surgically taken biopsies were collected immediately after the operation, cut into a 3 mm 3 sample and placed in a 4.5 ml cell. Four mL of hypertonic PBS buffer with NaCl, 4.5 g in 500 mL (Weestburg) was added to the cell along with the sample in the basket. Independently, the syringe plunger is then moved to the 15mL mark. The syringe is then connected to the extraction cell. The sample is then subjected to alternating pressure at a frequency of 1 Hz for one minute by moving the plunger in the syringe between the 15mL marking and 7.5 mL. During measurements carried out separately with a connected manometer, it appears that this change in volume corresponds to an effective pressure variation between 2 bar (compression state) and 1 bar bar (relaxed state) and 1 bar at the initial point when the piston rests. found at the 15mL mark. The procedure is repeated 3 times for each example. The extraction fluids were then stored at -20 ° C. The entire procedure lasted 3 minutes and the sample is returned for clinical evaluation. A similar protocol was applied to samples from tumors of cancer of the human breast, primary colorectal cancer, and liver and colon metastases and their adjacent normal counterparts.

2. Comparaison des résultats d'Immunohistochimie (IHC) sur échantillons ayant/n'ayant pas subit la méthode d'extraction selon l'invention. 2. Comparison of the Immunohistochemistry (IHC) results on samples which have / have not undergone the extraction method according to the invention.

Pour vérifier que la méthode selon l'invention ne change pas la morphologie et antigénicité des échantillons tissulaires, une analyse comparative de différents tissus humain et de souris ont été soumis à la méthode. Chaque échantillon de tissus a été divisé en deux; d'une part pour analyse pathologique de routine et d'autre part pour la méthode d'extraction selon l'invention aussi dénommée INV dans les figures 8 et 9. Les échantillons pour l'analyse de routine et pour la méthode d'extraction selon l'invention sont soumis à la même Immunohistochimie (IHC) selon le protocole connu de l'homme du métier et décrit par A.Bellahcene et V.Castronovo dans Am. J Pathol 1955 jan ; 146(1) : 95-100 .To verify that the method according to the invention does not change the morphology and antigenicity of the tissue samples, a comparative analysis of different human and mouse tissues were subjected to the method. Each tissue sample was divided in half; on the one hand for routine pathological analysis and on the other hand for the extraction method according to the invention also called INV in the figures 8 and 9 . The samples for the routine analysis and for the extraction method according to the invention are subjected to the same immunohistochemistry (IHC) according to the protocol known to those skilled in the art and described by A.Bellahcene and V. Castronovo in Am. J Pathol 1955 Jan; 146 (1): 95-100 .

Dans la figure 8, des échantillons de tumeurs colorectales primaires (n=10, panneau gauche) et des échantillons de foie métastasés (n=10, panneau droit) ont été soumis à l'analyse clinique de routine et à la méthode d'extraction selon l'invention suivi de l'analyse hematoxylin/eosin (H&E) et immunolabelling pour les marqueurs identifiés (MLH1, MSH2, MSH6, PMS2). L'évaluation quantitative pour chaque marqueur a été effectuée selon les méthodes connues de l'homme du métier et décrit par D.Waltregny & cie dans J Natl Cancer Inst. 1998 Jul 1;90(13):1000-8 .In the figure 8 , samples of primary colorectal tumors (n = 10, left panel) and samples of metastasized liver (n = 10, right panel) were subjected to the routine clinical analysis and to the extraction method according to the invention. followed by hematoxylin / eosin (H&E) analysis and immunolabelling for the identified markers (MLH1, MSH2, MSH6, PMS2). The quantitative evaluation for each marker was carried out according to methods known to those skilled in the art and described by D. Waltregny & cie in J Natl Cancer Inst. 1998 Jul 1; 90 (13): 1000-8 .

Des échantillons de 3 patients ont été collectés dans chaque cas. Chaque image IHC a été évaluée pour son intensité en utilisant l'échelle suivante (0 pour aucune trace, 1 pour faible, 2 pour modéré et 3 pour fort). Les tissus ont été également évalué pour l'étendue de leur positivité en utilisant l'échelle 1= 0-33%, 2= 33-66%, 3=66-100%. Les valeurs obtenues par chacune des deux évaluations sont multipliées pour donner le HIC score reproduit à la figure 8.Samples from 3 patients were collected in each case. Each IHC image was rated for its intensity using the following scale (0 for no trace, 1 for weak, 2 for moderate and 3 for strong). Tissues were also assessed for the extent of their positivity using the scale 1 = 0-33%, 2 = 33-66%, 3 = 66-100%. The values obtained by each of the two evaluations are multiplied to give the HIC score reproduced in the figure 8 .

Dans la figure 9, des échantillons de tissus de souris présentant un cancer du sein (n=3), un cancer colorectal (CRC ;n=3), un cancer du foie en métastase (CRC-LM ; n=3) ont été l'objet soit au test de routine soit à la méthode d'extraction suivi de l'analyse hematoxylin/eosin (H&E).In the figure 9 , tissue samples from mice with breast cancer (n = 3), colorectal cancer (CRC; n = 3), metastatic liver cancer (CRC-LM; n = 3) were either routine testing or extraction method followed by hematoxylin / eosin (H&E) analysis.

Comme illustré dans les figures 8 et 9 aucune différence significative n'a été mise en évidence concernant la structure du tissu (H&E) et l'intensité des marqueurs choisis.As illustrated in figures 8 and 9 no significant difference was demonstrated in tissue structure (H&E) and the intensity of the markers chosen.

3.utilisation du dispositif pour l'Identification de marqueurs protéiques et métaboliques au niveau du cancer du sein humain.3.use of the device for the identification of protein and metabolic markers in human breast cancer.

Dix prélèvements de cancer du sein et leur tissus non tumoral voisin ont été traités selon le protocole décrit à l'exemple 1 puis soumis au dispositif et méthode selon l'invention. Le liquide obtenu après application du dispositif d'extraction a alors été analysé par spectrométrie de masse pour la détection de protéines solubles et par Résonnance Magnétique Nucléaire pour la détection de métabolites. Les marqueurs identifiés à partir du liquide obtenu des tissus cancéreux ont alors été comparé à ceux des tissus normaux.Ten breast cancer samples and their neighboring non-tumor tissue were treated according to the protocol described in Example 1 and then subjected to the device and method according to the invention. The liquid obtained after application of the extraction device was then analyzed by mass spectrometry for the detection of soluble proteins and by Nuclear Magnetic Resonance for the detection of metabolites. The markers identified from the fluid obtained from cancerous tissue were then compared with those from normal tissue.

3.1 détection des marqueurs protéiques3.1 detection of protein markers

Parmi les protéines détectées dans les tissus cancéreux, on identifie par exemple les Periostin, comp, Ezrin, Raxidin, Versican, Tenascin. Ces protéines sont généralement présentes dans l'espace extracellulaire des tissus comme illustrés dans le tableau 1 ci-dessous reprenant des extraits de Pubmed : Tableau 1 : protéines identifiées dans les tissus du cancer du sein Periostin 49 Results on Pubmed COMP . 4 results on Pubmed • Protein involved in osteoblast adhésion. • Localization: extracellular space • Major component of ECM. • Tumor development • Secreted by fibroblasts • Metastases dissémination • Generally associated with poor patient outcome. Ezrin 114 results on Pubmed Radixin 63 results on Pubmed • Localization: plasma membrane Major association: • Localization: cell membrane, extracellular space • Ezrin-radixin-moesin (ERM) plys important role in the maintenance of cytoskeleton structure and cells movement. It also involved in breast cancer invasion and metastasis and may be a potential biomarker. This axis is regulated by miR-200c • EMT • Cell motility • Disease progression (ovarian cancer) • migration & invasion Versicon 36 results on Pubmed Tenascin 158 results on pumed • Localization: extracellular matrix Major association: Localization: extracellular space Function: • Cell motility Implicated in guidance of migrating neurons as well as axons during development Function in tumors: Stimulates angiogenesis • Cell differentiation • Cell growth • TGFB-1 induced Among the proteins detected in cancerous tissues, for example, Periostin, comp, Ezrin, Raxidin, Versican, Tenascin are identified. These proteins are generally present in the extracellular space of tissues as illustrated in Table 1 below, showing extracts from Pubmed: Table 1: Proteins Identified in Breast Cancer Tissues Periostin 49 Results on Pubmed COMP. 4 results on Pubmed • Protein involved in osteoblast adhesion. • Localization: extracellular space • Major component of ECM. • Tumor development • Secreted by fibroblasts • Dissemination metastases • Generally associated with poor patient outcome. Ezrin 114 results on Pubmed Radixin 63 results on Pubmed • Localization: plasma membrane Major association: • Localization: cell membrane, extracellular space • Ezrin-radixin-moesin (ERM) plys important role in the maintenance of cytoskeleton structure and cells movement. It also involved in breast cancer invasion and metastasis and may be a potential biomarker. This axis is regulated by miR-200c • EMT • Cell motility • Disease progression (ovarian cancer) • migration & invasion Versicon 36 results on Pubmed Tenascin 158 results on pumed • Localization: extracellular matrix Major association: Localization: extracellular space Function: • Cell motility Implicated in guidance of migrating neurons as well as axons during development Function in tumors: Stimulates angiogenesis • Cell differentiation • Cell growth • TGFB-1 induced

La figure 10 illustre le nombre de marqueurs protéiques identifiés dans le liquide obtenu suite au dispositif et à la méthode d'extraction selon l'invention. Les marqueurs identifiés à partir du liquide dans lequel un tissu cancéreux a été plongé, sont comparés à ceux extraits des tissus normaux.The figure 10 illustrates the number of protein markers identified in the liquid obtained following the device and the extraction method according to the invention. The markers identified from the liquid in which cancerous tissue has been immersed are compared with those extracted from normal tissue.

1032 protéines ont ainsi été identifiées à partir des tissus cancéreux et sont exprimées de manière différentielle (722 augmentées et 185 diminuées) dans la figure 10 1032 proteins have thus been identified from cancerous tissues and are differentially expressed (722 increased and 185 decreased) in the figure 10

3.2 détection des marqueurs métaboliques3.2 detection of metabolic markers

L'Ingenuity Patway Analysis a été utilisé pour l'interprétatin des données récoltées et leur analyse au moyen du logiciel IPA Core AnalysisThe Ingenuity Patway Analysis was used for the interpretation of the collected data and their analysis using the IPA Core Analysis software.

Dans la figure 11 sont illustrés les marqueurs métaboliques identifiés selon la méthode et le dispositif d'extraction selon l'invention. On assiste à une altération et une augmentation des métabolites dans le cas du cancer mammaire. L'alanine, la taurine et l'hypotaurine, la betaine, le glucose alanine sont les marqueurs ayant subi le plus important up-regulation comme le reflète la valeur de P.In the figure 11 The metabolic markers identified according to the method and the extraction device according to the invention are illustrated. There is an alteration and an increase in metabolites in breast cancer. Alanine, taurine and hypotaurine, betaine, glucose alanine are the markers that have undergone the most up-regulation as reflected in the value of P.

La valeur de P indique quelle molécule est associée significativement avec des moléculesde référence introduites et cela relativement à toutes les molécules fonctionnelles caractérisées. Le fold enrichment indique la signification statistique de la présence du métabolite dans l'échantillon.The value of P indicates which molecule is significantly associated with the introduced reference molecules and this relatively to all the functional molecules characterized. The fold enrichment indicates the statistical significance of the presence of the metabolite in the sample.

Claims (23)

  1. Method for extracting a marker, comprising:
    - introducing a sample into a removable support basket placed in the housing of a cell of an extraction device provided with a flexible membrane and
    - immersing the sample in a liquid previously introduced into the housing;
    - hermetically closing the extraction cell after immersion of the sample in the liquid and
    - applying a pressure cycle on the flexible membrane for a period of 1 to 5 minutes, at a frequency of between 1 and 2 Hz; then
    - totally or partially recovering the liquid immersing the sample after the pressure cycle, and detecting the markers present in the physiological liquid;
    characterized in that:
    the pressure applied on the membrane is between 2 and 5 bar.
  2. Method according to Claim 1, characterized in that the duration of the pressure cycle is 3 minutes.
  3. Method according to either one of the preceding claims, characterized in that the sample is a biological tissue sample and the liquid is a physiological liquid for the extraction of biomarkers.
  4. Method according to Claim 3, characterized in that the biological tissue sample is extracted from a cancerous tumor.
  5. Method according to either one of Claims 3 and 4, characterized in that the physiological liquid is a PBS buffer with 9 g/l NaCl.
  6. Device (1) for extracting a marker, comprising:
    - an extraction cell (3) provided with a housing (12) for receiving a removable support basket (5), said basket (5) being configured to immerse a sample (6) in a liquid previously introduced into the housing (12);
    - a cavity (7) separated from the housing of the extraction cell (3) by means of a flexible membrane (8), allowing a compression or a dilation of the liquid; and
    - a leaktight cap (2) placed on the extraction cell (3) for attaching the removable support basket (5) in the housing (12).
  7. Device according to Claim 6, characterized in that the leaktight cap comprises two portions, an upper portion or Luer Lock cap (2) placed on a lower part (4) configured to maintain the leaktightness of the extraction cell during the pressure cycle and to allow the extraction of the liquid at the end of the cycle.
  8. Device according to either one of Claims 6 and 7, characterized in that it also comprises a piston (11) cylinder (10) connected to the cavity (7) at the orifice (13) .
  9. Device according to Claim 8, characterized in that the cavity (7) and the volume of the cylinder (10) above the piston are filled with air.
  10. Device according to any one of Claims 6 to 9, characterized in that the cavity (7) also comprises at least one stiffener (9).
  11. Device according to Claim 10, characterized in that at least two stiffeners are placed in a star pattern around the cylinder (10) connection orifice (13).
  12. Device according to any one of Claims 6 to 11, characterized in that the support basket (5) is provided with vertical ribs allowing the immersion of the sample in the liquid.
  13. Device according to the preceding Claim 12, characterized in that the ribs of the support basket (5) must have a width suitable for the size of the sample.
  14. Device according to any one of Claims 6 to 13, characterized in that the support basket (5) is made of plastic, preferably polypropylene.
  15. Device according to any one of Claims 6 to 14, characterized in that the flexibility of the membrane (8) allows a degree of deformation of 1 to 2 times its surface area.
  16. Device according to any one of the preceding Claims 8 to 15, wherein the cylinder (10) is vertically connected to the extrusion cell (3).
  17. Device according to any one of the preceding Claims 8 to 16, wherein the cylinder (10) provided with the piston (11) is a disposable syringe.
  18. Extraction device according to any one of the preceding Claims 6 to 17, characterized in that the sample is a biological tissue sample and the liquid is a physiological liquid for the extraction of biomarkers.
  19. Device for extracting a biomarker according to Claim 18, for the early diagnosis of cancer at the primary stage.
  20. Device for extracting a biomarker according to Claim 18, for cancer treatment.
  21. Use of the device according to Claim 18, for detecting molecular metabolic and protein biomarkers from a solid biopsy.
  22. Apparatus comprising the device for extracting markers according to any one of Claims 8 to 18, and also a means for actuating the travel of the piston.
  23. Apparatus according to Claim 22, characterized in that the means for actuating the travel of the piston is a jack.
EP18726776.0A 2017-09-06 2018-05-19 Non-destructive sampling device for extraction of a marker Active EP3679374B1 (en)

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BE2017/5633A BE1025528B1 (en) 2017-09-06 2017-09-06 NON-DESTRUCTIVE SAMPLE DEVICE FOR EXTRACTING MARKERS
PCT/EP2018/063244 WO2019048087A1 (en) 2017-09-06 2018-05-19 Non-destructive sampling device for extraction of a marker

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EP3679374B1 true EP3679374B1 (en) 2021-06-23

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JP4559150B2 (en) * 2004-07-28 2010-10-06 テルモ株式会社 Syringe pump
JP2015139398A (en) * 2014-01-28 2015-08-03 株式会社ライフテック Apparatus and method for nucleic acid extraction
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BE1025528B1 (en) 2019-04-05
US20210072127A1 (en) 2021-03-11
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WO2019048087A1 (en) 2019-03-14
JP2020532729A (en) 2020-11-12

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